Device and method for extraction and analysis of nucleic acids from biological samples

ABSTRACT

Device and methods for extracting and analyzing nucleic acids from biological samples.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/768,076, filed Jun. 25, 2007, which claims the benefit of U.S.Provisional Application No. 60/816,577, filed Jun. 26, 2006, and U.S.Provisional Application No. 60/910,609, filed Apr. 6, 2007. Eachapplication is expressly incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Platelets are a component of blood comprised of anucleate megakaryocytefragments that circulate in the blood for about 10 days. When separatedas a component of whole blood, platelets are routinely concentrated,re-suspended in plasma and/or platelet additive solutions, leukoreducedby passage through a filtration device and stored in platelet storagebags which are kept on flatbed agitators for 5 to 7 days at atemperature of 22° C. The relative DNA content in whole blood platelets(WBP) or leukoreduced/apheresis platelets varies considerably.

Microbial contamination of blood transfusion products is a major medicalproblem. Blood banks are faced with a great challenge in testing eachplatelet bag for microbial contamination prior to release for infusioninto a patient. Currently contaminated platelets are often infused intopatients, and the physician is notified subsequently that the plateletswere contaminated as the culture results become available. Under theAmerican Association of Blood Banks (A.A.B.B.) standard 5.1.5.1, bloodbanks or transfusion services are instructed to have methods to limitand detect bacterial contamination in all platelet concentrates. NucleicAcid Testing (NAT) technology would allow testing of bacteriallycontaminated units to be detected rapidly after the first day ofstorage, thus ensuring a safe transfusion by eliminating the possibilityof contaminated platelets.

With the successful implementation of stringent control measures, theestimated risk of infection by well known viral pathogens such as HIVand HCV has fallen below 1 per 1 to 2 million transfusion units. Thiswas achieved in part through the use of nucleic acid based testingperformed on pooled products composed of 16 to 24 units. In contrast,the risk of transfusion with bacterially contaminated platelets may beas a high as 1 in 1,000 units, with perhaps 10% to 25% of such incidentsresulting in adverse effects on patients. A recent study on themicrobiological safety of transfusions concluded that a rapid test formicrobial contamination in platelets with a detection limit of 10³ or10⁴ organisms/ml is a desirable target.

Vogelstein and Gillespie described the purification of DNA using glassas a DNA binding surface in the presence of high concentrations ofchaotropic salts, for example, GuSCN, GuHCl, NaI, or NaClO₄. The bindingof sequence specific DNA probes to glass microscope slides for use inDNA microarrays was also previously described. The slides were not,however, used for capture of genomic DNA from samples. One advantage ofthe flat glass microscope slides is the reproducibility of the glassproduct. Glass microscope slides are made by Erie Scientific(Portsmouth, N.H.) from soda lime glass. Thin glass sheets are drawnfrom the molten glass using the “electroverre” process and the materialis known as “Swiss glass.” Glass microscope slides are cut from largethin (1 mm thick) sheets. Glass slides have been used for decades inmedical diagnostics and the material is very uniform.

U.S. Pat. No. 5,234,809 describes a one-step process for purification ofnucleic acids from complex material such as body fluids or otherbiological starting materials. In the method, a process is described forisolating nucleic acids from starting material comprising mixing thestarting material, a chaotropic substance, and a nucleic acid solidphase such as glass cuvettes, separating the solid phase with thenucleic acid bound to it from the liquid, and washing the solid phasenucleic acid complexes. However, it is well known that such a one-stepprocess often cannot be used to purify a significant amount of the totalnucleic acids available in the starting mixture. This can bedemonstrated using even known quantities of nucleic acids added to acomplex biological fluid—plasma—prior to purification over a wellunderstood commercial DNA purification kit using processes such as thosedescribed in U.S. Pat. No. 5,234,809.

For extraction of nucleic acids from blood or blood products,proteolysis of the sample is commonly carried out prior to purificationof the DNA. Enzymes such as proteinase K, lysostaphin, or other similarenzymes that are either expressed in organisms such as Esherichia colior Pichia pastoris and purified, or purified from other sources, can beused for proteolysis.

NAT is a powerful analytical tool for determining the presence ofgenetic material (DNA or RNA) in biological samples. For example,polymerase chain reaction (PCR) can be used to detect trace microbialcontamination in sterile systems and to find pathogenic gene sequencesin the human cells. In blood banks, NAT is commonly used to detect thepresence of viral contamination (HIV, HBV, HCV) in blood products.

It has been previously shown using a NAT protocol that the sampling ofpooled platelets after one day of storage affords the accurate detectionof most bacterial species in spiked platelet concentrates (PCs) atdetection levels that equal or exceed the culture systems currently inuse for bacterial testing of PCs. The NAT protocol provides an inherentadvantage in providing results within a short amount of time in a closedsterile device that would allow for the clinical determination of thelevel of microbial contamination within a sample.

SUMMARY OF THE INVENTION

The present invention provides a device and method for extracting andanalyzing nucleic acids from biological samples. The method and deviceare useful for determining the quantity of nucleic acids in a sample.

In one aspect, the invention provides a fluorescence method fordetermining the quantity of nucleic acids in a sample. In oneembodiment, the method includes:

(a) introducing a sample containing cells in a liquid medium into afirst chamber of a vessel, the vessel comprising the first chamber and asecond chamber, wherein the first chamber is in liquid communicationwith the second chamber and wherein the second chamber has at least aportion of one surface effective for binding nucleic acids;

(b) lysing the cells to provide nucleic acids in the liquid medium;

(c) transferring at least a portion of the liquid medium from the firstchamber into the second chamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to the surface of the second chamber effective for bindingnucleic acids to provide isolated nucleic acids;

(e) contacting the isolated nucleic acids with a fluorescent compoundhaving a fluorescence intensity dependent on the concentration ofnucleic acids; and

(f) measuring the fluorescence of the fluorescent compound to determinethe quantity of isolated nucleic acids.

In one embodiment, the sample is blood or a blood product.

In another aspect of the invention, a method for amplifying andquantifying the amount of nucleic acids in a sample is provided. In oneembodiment, the method includes:

(a) introducing a sample containing cells in a liquid medium into afirst chamber of a vessel, the vessel comprising the first chamber and asecond chamber, wherein the first chamber is in liquid communicationwith the second chamber and wherein the second chamber has at least aportion of one surface effective for binding nucleic acids;

(b) lysing the cells to provide nucleic acids in the liquid medium;

(c) transferring at least a portion of the liquid medium from the firstchamber into the second chamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to a surface of the second chamber effective for bindingnucleic acids, to provide isolated nucleic acids;

(e) releasing the isolated nucleic acids from the surface of the secondchamber effective for binding nucleic acids by contacting the isolatednucleic acids with a buffer solution;

(f) treating the isolated nucleic acids with and a nucleic acidamplification reaction mixture under conditions for amplifying theisolated nucleic acids to provide amplified nucleic acids;

(g) contacting the amplified nucleic acids with a fluorescent compoundhaving a fluorescence intensity dependent on the concentration ofnucleic acids; and

(h) measuring the fluorescence of the fluorescent compound to determinethe quantity of amplified nucleic acids.

In one embodiment the method includes repeating steps (f) and (h) apre-determined number of times to determine the amount of amplifiednucleic acids.

In one embodiment, the method includes contacting the isolated nucleicacids with a fluorescent compound having a fluorescence intensitydependent on the concentration of nucleic acids and measuring thefluorescence of the fluorescent compound to determine the quantity ofisolated nucleic acids prior to step (f).

In another aspect of the invention, a fluidic system including a vesselis provided. The vessel includes:

(a) a fluid inlet port;

(b) a first chamber in fluid communication with the inlet port;

(c) a second chamber in fluid communication with the first chamber,wherein the second chamber has at least one glass surface;

(d) a first channel that connects the first chamber and the secondchamber;

(e) a fluid outlet port; and

(f) a second channel that connects the second chamber to the fluidoutlet port.

In one embodiment the fluidic system includes a device for measuring thefluorescent intensity of a fluorescent compound.

In another aspect, the invention provides a method for determining themicrobial content of a blood product including:

(a) introducing a sample of a blood product containing cells in a liquidmedium into a first chamber of a vessel, the vessel comprising the firstchamber and a second chamber, wherein the first chamber is in liquidcommunication with the second chamber, and wherein the second chamberhas at least a portion of one surface effective for binding nucleicacids;

(b) contacting the cells in the liquid medium with a lysis buffer toprovide nucleic acids in the liquid medium;

(c) transporting at least a portion of the liquid medium into the secondchamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to the surface of the second chamber effective for bindingnucleic acids to provide isolated nucleic acids;

(e) releasing the isolated nucleic acids from the surface of the secondchamber effective for binding nucleic acids by contacting the isolatednucleic acids with an elution buffer to provide released nucleic acids;

(f) contacting the released nucleic acids with a fluorescent compoundhaving a fluorescence intensity dependent on the concentration ofnucleic acids and a nucleic acid amplification reaction mixture;

(g) measuring the fluorescence of the fluorescent compound to determinethe quantity of released nucleic acids;

(h) treating the released nucleic acids and the nucleic acidamplification reaction mixture under conditions for amplifying theisolated nucleic acids by polymerase chain reaction to provide amplifiednucleic acids; and

(i) measuring the fluorescence of the fluorescent compound to determinethe quantity of amplified nucleic acids.

In one embodiment the method includes repeating steps (h) and (i) apre-determined number of times to determine the amount of amplifiednucleic acids.

In another aspect the invention provides a method for quantifying andamplifying the amount of nucleic acids in a sample. The method includes:

(a) contacting nucleic acids from a sample with a nucleic acidamplification reaction mixture that comprises a first fluorescentcompound having a first fluorescence emission maximum and a secondfluorescent compound having a second fluorescence emission maximum,wherein the first and second fluorescence emission maxima are different,each having a fluorescence intensity dependent on the concentration ofnucleic acids;

(b) measuring the fluorescence of the first fluorescent compound todetermine the amount of nucleic acids;

(c) treating the nucleic acids and the nucleic acid amplificationreaction mixture under conditions for amplifying the nucleic acids toprovide amplified nucleic acids; and

(d) measuring the fluorescence of the second fluorescent compound todetermine the amount of amplified nucleic acids.

In one embodiment, the nucleic acid amplification reaction mixturefurther comprises a third fluorescent compound having a thirdfluorescence emission maximum and a fluorescence intensity dependent onthe concentration of amplified nucleic acids, wherein the thirdfluorescence emission maximum is different from the first and secondfluorescence emission maxima.

In another aspect, the invention provides a composition including anucleic acid amplification reaction mixture and a fluorescent compound.The fluorescent compound has a fluorescence intensity dependent on theconcentration of nucleic acids and has a fluorescent emission maximumless than about 500 nm.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a schematic illustration of a representative fluidic device ofthe invention;

FIG. 2 is a graph comparing the amount of DNA recovered from variouslysized DNA fractions by two different methods;

FIG. 3 is a graph comparing the amount of DNA recovered by arepresentative method of the invention using different reagentformulations;

FIG. 4 is a graph showing the amount of DNA recovered as a function ofbinding time using a representative method of the invention;

FIG. 5 is a graph showing the amount of DNA recovered as a function ofelution time using a representative method of the invention;

FIG. 6 is a graph comparing the amount of DNA recovered from slidessubjected to different temperatures during binding and elution;

FIG. 7 is a graph comparing the amount of DNA recovered from slides thatwere pre-treated using various cleaning protocols;

FIG. 8 is a graph comparing the amount of DNA recovered from differentslides under similar conditions;

FIG. 9 is a graph comparing the amount of DNA recovered using twodifferent methods;

FIG. 10 is a graph comparing the amount of DNA recovered followingpost-lysis sonication for different types of cell samples;

FIG. 11 is a graph comparing the amount of DNA released after lysis fromsonicated and unsonicated bacterial cell cultures;

FIG. 12 is a graph comparing the amount of DNA recovered from sonicatedand unsonicated bacterial cell cultures using a representative method ofthe invention;

FIG. 13 is a graph showing the amount of DNA recovered followingdifferent sonication times;

FIG. 14 is a graph comparing the amount of DNA recovered from differentconcentrations of PCs using two different methods;

FIG. 15 is a graph comparing the amount of DNA recovered to the numberof glass slide surfaces used to isolate DNA using a representativemethod of the invention;

FIG. 16 is a graph comparing the amount of DNA recovered from differentsamples as a function of the number of slides used to isolate DNA usinga representative method of the invention;

FIG. 17 is a graph showing real-time PCR C(t) values for PC samples withdifferent platelet concentrations;

FIG. 18 is a graph showing real-time PCR C(t) values for samples withdifferent platelet concentrations;

FIG. 19 is a graph showing real-time PCR C(t) values for PC samples withand without bacterial contamination;

FIG. 20A is an exploded view of the layers of a representative fluidicdevice of the invention;

FIG. 20B is a schematic view of the assembled layers of a representativefluidic device of the invention having a glass surface on each side;

FIGS. 21A-21F are schematic illustrations depicting the transport offluids through a representative fluidic device of the invention; and

FIGS. 22A and 22B are schematic illustrations of a representative deviceand method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a device and method for extracting andanalyzing nucleic acids from biological samples. The device and methodare useful for determining the quantity of nucleic acids in a biologicalsample. The quantity of nucleic acids is determined by contacting thenucleic acids with a fluorescent compound having a fluorescenceintensity dependent on the concentration of nucleic acids and measuringthe fluorescence of the fluorescent compound. The device and method ofthe invention are useful for measuring the quantity of nucleic acids ina blood sample or blood product (e.g., platelets) and determiningwhether the sample is contaminated with bacterial pathogens.

In one aspect, the invention provides a fluorescence method fordetermining the quantity of nucleic acids in a sample. In oneembodiment, the method includes:

(a) introducing a sample containing cells in a liquid medium into afirst chamber of a vessel, the vessel comprising the first chamber and asecond chamber, wherein the first chamber is in liquid communicationwith the second chamber and wherein the second chamber has at least aportion of one surface effective for binding nucleic acids;

(b) lysing the cells to provide nucleic acids in the liquid medium;

(c) transferring at least a portion of the liquid medium from the firstchamber into the second chamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to the surface of the second chamber effective for bindingnucleic acids to provide isolated nucleic acids;

(e) contacting the isolated nucleic acids with a fluorescent compoundhaving a fluorescence intensity dependent on the concentration ofnucleic acids; and

(f) measuring the fluorescence of the fluorescent compound to determinethe quantity of isolated nucleic acids.

Nucleic acids including deoxyribonucleic acids (DNA) or ribonucleicacids (RNA) can be extracted and analyzed by the method. In oneembodiment, the sample is blood or a blood product (e.g., platelets) andthe nucleic acids that are extracted and analyzed are those fromcontaminant bacterial pathogens in the blood or blood product.

The vessel includes an inlet to the first chamber (lysis chamber), afirst channel that connects the first chamber to the second chamber(extraction chamber), an outlet, and a second channel that connects thesecond chamber to the outlet. A representative vessel useful forcarrying out the method of the invention is shown in FIG. 1. Referringto FIG. 1, the representative vessel 10 includes inlet 100, firstchamber 200, first channel 300, second chamber 400, second channel 500,and outlet 600. Glass cover 450 defines at least one surface of secondchamber 400. In certain embodiments, second chamber 400 is defined bytwo glass slides (i.e., floor and ceiling of the chamber, see FIG. 20B).The vessel is described below. As used herein, the terms “card” and “DNAcard” refer to the vessel of the invention.

In one embodiment, the vessel is sealable. Sealing the vessel reducesthe likelihood of environmental contamination of the sample, and reducesthe likelihood that a sample handler will be exposed to a contaminatedsample.

One feature of the device of the invention is that cells from the sampleare lysed in the same vessel in which nucleic acid extraction andanalysis is performed. In one embodiment, lysing the cells of the samplecomprises contacting the cells with a chaotropic salt solution. Lysingthe cells can further include sonicating or mechanically disrupting thecells in the sample in the first chamber.

In the methods, transferring the liquid medium from the first chamberinto the second chamber may include rotating the vessel so that theliquid medium is transferred into the second chamber by gravity.Transferring the liquid medium from the first chamber into the secondchamber may also include pumping the liquid medium from the firstchamber into the second chamber.

Nucleic acids are extracted from the liquid medium in the second chamberby binding to a surface effective to bind nucleic acids to provideisolated nucleic acids. The second chamber includes at least one surfacehaving at least one portion that is effective for binding nucleic acids.In one embodiment, the surface that is effective for binding nucleicacids is a glass surface (flat glass surface). See, for example, surface450 in FIG. 1. In another embodiment, the surface that is effective forbinding nucleic acids is a surface bearing an immobilized agent that iseffective for binding nucleic acids (e.g., immobilized minor groovebinder or intercalator).

To determine the quantity of isolated nucleic acids, the nucleic acidsare contacted with a fluorescent compound having a fluorescent intensitydependent on the concentration of nucleic acids and measuring thefluorescence of the fluorescent compound. Fluorescent compounds having afluorescent intensity dependent on the concentration of nucleic acidsare fluorescent compounds that exhibit a change in fluorescenceintensity in the presence of nucleic acids. Useful fluorescent compoundsinclude those compounds whose intensity increases in the presence ofnucleic acids. Other useful fluorescent compounds includeoligonucleotide probes labeled with both a fluorophore and a quencherand that cleave during nucleic acid amplification releasing fluorophoreindicating the presence of specific nucleic acids. Representativefluorescent compounds include fluorogenic minor groove binder agentssuch as bis-benzimide compounds and intercalating fluorogenic agentssuch as ethidium bromide. In one embodiment, the fluorescent compound isimmobilized in the second chamber. Methods for immobilizing thefluorescent compound in the second chamber and useful fluorescentcompounds are described below and in US 2006/0166223 A1, incorporatedherein by reference in its entirety. In another embodiment, the methodincludes releasing the isolated nucleic acids from the surface of thesecond chamber effective for binding nucleic acids by contacting theisolated nucleic acids with a buffer solution before measuring thefluorescence of the fluorescent compound. In this method, the buffersolution may include the fluorescent compound.

The vessel of the invention allows for the interrogation of the secondchamber by fluorescence by having at least a portion of the chambersuitable for transmitting excitation energy to the fluorescent compoundsin the second chamber and for transmitting fluorescence emissionintensity from the compounds in the second chamber.

In one embodiment, the method further includes purifying the isolatednucleic acids by contacting the isolated nucleic acids with a chaotropicsalt solution.

For use in analyzing nucleic acids present in platelets stored in aplatelet storage bag, the vessel can be attached directly to the storagebag without contaminating the contents of the bag. In practice, thevessel can be directly attached bag's sampling pouch followed bysterilization of the bag with ethylene oxide using standard conditions.The bag's tubing configuration allows platelet-rich plasma (PRP) to bedirectly inputted to the vessel with no requirement for aseptic samplehandling. For identification control, the bag and card can have the samebar code (or other identifier) for tracking purposes. For example, a PRPsample (about 5 ml) can be drained into a sample bag that issealed/removed from the storage bag. The removed sample would then betested by hematology and viral NAT as usual, optionally cultured toenhance possible bacterial contamination, and then a PRP sample (about0.2 ml) is transferred from the sample bag to the vessel for analysis.

In certain embodiments, the devices and methods of the invention use aglass surface (e.g., a flat glass surface such as a glass slide, seeFIG. 1, reference numeral 450) to extract and isolate nucleic acids froma liquid medium. The devices and methods of the invention that use theglass surface nucleic acid capture are referred to herein as the “glassslide” system or method.

Recovery of purified DNA using the glass slide system used in the methodand device of the invention was compared to the standard methoddeveloped by Qiagen. Recovery of high molecular weight DNA from calfthymus was compared with DNA for bacteriophage lambda using either theglass slide method or the Qiagen Blood Mini Kit. For the Qiagen method,purified DNA was suspended in Tris-EDTA (TE) buffer followed by additionof Qiagen AL Buffer (lysis buffer) and pure ethanol as directed in thepackage insert, except that protease was not included. Samples wereapplied to the sample purification columns, washed with wash buffers 1and 2, and finally eluted with 100 ul TE buffer. For the glass slideprotocol, samples were prepared as for the Qiagen method except that 300ul of the sample was layered onto a glass microscope slide. After 20minutes of incubation, the slide was washed with Qiagen wash buffers 1and 2. DNA was eluted from the glass slide with 200 ul of TE buffer. Allsamples were quantified using the PicoGreen assay (Invitrogen). Theresults of this experiment were as follows. Using the Qiagen method,recovery of lambda DNA was 94%, and recovery of calf thymus DNA was 20%.Using the glass slide method, recovery of lambda DNA was 20%, andrecovery of calf thymus DNA was 20%. The results indicate that eventhough the recovery of lambda DNA is higher in the Qiagen method,recovery of high molecular weight calf thymus DNA is equivalent betweenthe two systems.

The relatively poor yield of high molecular weight calf thymus DNA fromboth systems suggests that some property of either the samplepreparation protocol or of the sample itself affects the recovery ofDNA. One possibility is the size of the DNA substrate used in thebinding protocol. To explore this possibility, salmon sperm DNA waspurchased from Sigma, suspended in TE buffer, and sonicated for variouslengths of time to fractionate or break down the size of the DNA. Thesize of individual fractions was assessed using agarose gelelectrophoresis. Sizes of fractions chosen for binding included (1)unsonicated DNA (>50 Kb); (2) fraction 3 with an average size of >8 Kb;(3) fraction 4 with an average size of 2 to 8 Kb; and (4) fraction 5(35% power) with an average size of less than 8 Kb with a lower rangebelow that of fraction 4. The results of an experiment showing theeffect of sonication on the recovery of DNA using the Qiagen method andglass slide method is shown in FIG. 2.

Referring to FIG. 2, fraction 1 corresponds to purified lambda DNA (2.5ug input). Fraction 2 is unsonicated DNA, fraction 3 is the >8 Kbsonicated fraction, fraction 4 is the 2 to 8 Kb sonicated fraction, andfraction 5 is less than the sonicated >8 Kb fraction. The results showthat the recovery of DNA from glass slides is overall less than that ofthe Qiagen method. In general, with the purified DNA, recovery of DNA isrelatively insensitive to the size of the DNA offered to either glassslides or to the Qiagen system. In particular, comparison of unsonicatedDNA with fraction 3 DNA (>8 Kb) shows little dependence of size onrecovery. This implies that there is little difference in recoverybetween unsonicated DNA and an 8 Kb size sonicated DNA fraction. Aslight effect is seen in the lower sized DNA.

Reagents were developed and compared to the commercially availableQiagen reagents. A set of recipes was used that included alysis/extraction buffer containing 5M guanidine thiocyanate, 20 mM EDTA,and 1% Triton in 0.1M Tris pH 6.4. Wash buffer 1 had the samecomposition except that it lacked the Triton. Wash buffer 2 had 10 mMTris pH 7.4, 2.5 mM EDTA, 50 mM NaCl, and 50% ethanol. The elutionbuffer contained 10 mM Tris pH 8.0 and 1 mM EDTA (TE buffer).

In contrast to the Qiagen formulations for AL Buffer (lysis buffer)which uses an addition of ethanol, no ethanol dilution is required forthe reagents useful in the methods of the invention. Thelysis/extraction buffer may be used with no further additions. If aprotease is required for lysis, the lysis/extraction buffer is diluted.To assess the effectiveness of the reagent formulations, a slide bindingassay was run using 2.5 ug of sonicated DNA (>8 Kb size fraction) tocompare DNA yield using the new reagents to DNA yield using the Qiagenreagents. The results are shown in FIG. 3. In this experiment, variouscombinations of reagents were tested. Referring to FIG. 3, the fullreagent set is represented by the designation “+Triton”. Triton was leftout (“−Triton”), ethanol was added along with triton to mimic the Qiagenreagent set (“+Triton+EtOH”), and again triton was excluded(“−Triton+EtOH”). Although some minor differences were obtained in thisexperiment, none of the reagent sets varied significantly from theQiagen reagent set. This indicated that the defined reagent set caneffectively substitute for the Qiagen reagent set in the DNA bindingassay.

To determine the optimum binding and release times for glass slides, thetime needed for DNA adsorption was measured. First, 500 ng ofunsonicated DNA was suspended in 300 ul of lysis/extraction buffer andapplied to a glass slide for time periods ranging from 0 to 35 minutes.Binding was halted by centrifugation of the DNA binding mix from theglass slide. The glass slides were rinsed in wash buffers 1 and 2. DNAwas eluted from the glass slides using 200 ul of TE buffer. The resultsare shown in FIG. 4. Referring to FIG. 4, binding increases over theentire time period of the experiment from 5 minute to 30 minutes.Binding is starting to level off at 30 minutes although a slightincrease is still seen at this point. However, over 85% of binding hasoccurred in the first 20 minutes of the binding curve. Previous data wascollected with a 10 minute binding time. The percent recovery increasesover this 10 minute time period from about 16% to over 24%. Therefore, atime period for binding of at least 20 minutes is preferred.

The kinetics of elution of high molecular weight DNA was similarlyfollowed. A set of slides was prepared in which unsonicated salmon spermDNA was bound. The elution step was initiated by layering on TE bufferfor the indicated periods of time. The time course was stopped bycentrifugation of individual slides. The results are presented in FIG.5. Referring to FIG. 5, the results show that the most of the DNA hasbeen eluted at only 5 minutes into the time course.

Another factor that may influence the slide binding assay is theapplication of heat either to the binding phase, or elution, or both.Referring to FIG. 6, the effect of temperature was tested in a standardslide binding assay in which 500 ng of unsonicated DNA was suspended in300 ul of lysis/extraction buffer and applied to slides. Two slides wereincubated at 56° C. for 15 minutes (to minimize evaporation) while twoslides were incubated at room temperature. Slides were then rinsed inwash buffers 1 and 2. DNA was eluted in 200 ul TE buffer either at 56°C. or at room temperature. Referring again to FIG. 6, Slide 1 had roomtemperature binding and elution. Slide 2 had room temperature bindingand 56° C. elution. Slide 3 had 56° C. binding and room temperatureelution. Slide 4 had 56° C. binding and 56° C. elution. The results inFIG. 6 show that a reproducible effect of elution at 56° C. is observedthat ranges from 5% to 20% increase in DNA recovered. Heating at thebinding stage of the protocol greatly decreases the amount of DNA thatone can expect to recover from glass slides.

The need for extra cleaning of slides and slide-to-slide reproducibilitywas also studied to determine whether the cleanliness of the glassslides has an influence on binding and recovery of DNA. The yield ofrecovered DNA was thought to be increased if the glass slides werecleaned thoroughly with acid or base. Referring to FIG. 7, glass slideswere washed in either 1N nitric acid or sodium hydroxide and then testedin the slide binding assay. This experiment was done with DNA at both100 and 500 ng total input. Both the acid and base washed slides werefound to have a greatly decreased amount of DNA recovered as opposed tothe standard slides that were not further cleaned. The results shown inFIG. 7 suggest that further cleaning of glass slides is detrimental toefficient slide binding.

An assay was also performed in which the slide-to-slide variation wasmeasured. Referring to FIG. 8, a series of slides were prepared in which500 ng of total input unsonicated DNA was bound to each slide with abinding time of 30 minutes. Slides were rinsed in wash buffer 1 and 2,and DNA was eluted with 200 ul TE buffer. A total of 16 slides weretested. One representative set is shown in FIG. 8. The standard glassslide surface gave consistent DNA recovery. Out of 16 slides, one slidewas very low and was discarded from the analysis. Of the remainingslides, the standard deviation was 10%. The average yield on the slidesin FIG. 8 is about 300 ng of DNA, meaning that the average percent yieldis approximately 60%.

DNA recovery on glass slides is reduced, but not destroyed, when the DNAis suspended in biological media. In the presence of a physiologicalamount of human serum albumin (HSA), the DNA recovery is reduced 32%.When 25% plasma is added to the DNA/lysis buffer mix, recovery isreduced 75%.

Because several sources of exogenous nucleic acids found in thebiological samples may be recalcitrant to a particular lysis buffer andproteolytic digestion, other methods were developed to carry out morecomplete lysis of such samples concomitantly or at other points duringthe preparation. To further demonstrate the inability to recover nucleicacids comprised of genomic DNA efficiently from a complex mixture ofbacteria (Staphylococcus aureus or Escherichia coli at variousconcentrations) in human plasma and platelet concentrates using themethods described in U.S. Pat. No. 5,234,809, the following experimentwas performed. Referring to FIG. 9, the novel combination of heating andof sonic probe sonication of the lysates for various times (from 0-20seconds) prior to adsorption of the nucleic acid onto the glass slideswas performed. The results demonstrate a substantial increase inrecovered nucleic acid with increased sonication times. FIGS. 9 and 10shows the percentage recoveries of such nucleic acids from plasmafollowing the methods taught in the Qiagen Blood Mini Kit and thosedescribed herein, and in both cases the recovered amounts are low (<12%of offered).

U.S. Pat. No. 6,235,501 describes a method for the isolation of highmolecular weight DNA from biological samples. Controlled oscillatorymechanical energy is applied to the sample for short periods of time(5-60 seconds) to lyse the sample and release the DNA. A sphericalparticle is used for applying the mechanical energy. In the practice ofthe present invention, mechanical disruption of the biological samplematerials uses a sonicator probe, a piezobuzzer device, or anothersimilar device capable of introducing high frequency resonant vibrationsto the sample through a chamber wall of the DNA card. The shearing ofDNA in the methods of the invention significantly increases the totalamount of DNA released from the sample that is purified from the glasssurfaces compared to other methods well known to those who practice inthe field of nucleic acid extraction. In addition, to facilitate thelysis and inactivation of pathogenic organisms within the varioussamples as described above, the sample will also be heated totemperatures up to 95° C. for a period of time of 2-10 minutes in thepresence of various combinations of reagents and, if needed,concomitantly with the other disruptive methods such as described above.

Referring to FIG. 11, the total input DNA from a complex startingmaterial comprised of lysate was measured after mixing plasma and aknown quantity of bacterial cells (Staphylococcus aureus). FIG. 11 showsequivalent release of genomic DNA from the samples into each lysatefollowing probe sonication for 90 seconds and the 95° C. 10 minuteheating steps. FIG. 12 shows increased recovery of said genomic DNA fromthe samples following probe sonication for 90 seconds and the heatingstep as described above using the methods of the present invention.

FIG. 13 is a graph showing the amount of DNA recovered followingdifferent sonication times. Referring to FIG. 13, with regard to thelevel of sonication or similar mechanical disruption needed for therecovery of nucleic acids from biological materials using the protocolsdescribed herein, the optimum time of probe sonication at about 350 MHzcarried out with a Branson Sonifier on a lysate in an Eppendorf tube wasdetermined in the following experiment. Several samples containing 200ul of an outdated platelet concentrate were extracted and exposed tovarious time periods of continuous sonication. For the longer timeperiods of sonication (>30 seconds) the samples were briefly cooled onan ice slurry following one 30 second pulse. The total DNA recoveredfrom the glass slides following elution is shown in the graph in FIG. 13as quantified with the PicoGreen assay as per the manufacturer'sinstructions (Invitrogen Inc.) in a BioTek plate reader using theappropriate filter sets. Based on these results, the time of sonicationas carried out with the Branson probe sonicator as described aboveshould normally be about 90 seconds and applied in three separate 30second pulses.

FIG. 14 is a graph comparing the amount of DNA recovered from samplescontaining different PC concentrations using two different methods.Referring to FIG. 14, experiments were conducted to compare theperformance of the glass slide purification system with the Qiagen BloodMini Kit, a commercially available DNA purification system. In thisexperiment, lysates containing DNA extracted from 200 ul of platelets(prepared as described below) were split evenly and dispersed ontoeither a Qiagen column or a single glass slide, respectively. For theQiagen column preparations the samples were then washed with washbuffers 1 and 2, and finally eluted with 200 ul of TE. After 20 minutesof incubation, the slides were washed with wash buffers 1 and 2. DNA waseluted from the glass slides with 200 ul of TE. All samples werequantified using the PicoGreen assay (Invitrogen) as per themanufacturer's instructions. The results of this experiment are shown inFIG. 14.

FIG. 15 is a graph comparing the amount of DNA recovered to the numberof glass slide surfaces used to isolate DNA using the method of theinvention. Referring to FIG. 15, to demonstrate that the levels of DNArecovered from these samples are directly related to the total area ofthe glass slides a titration experiment was carried out. Briefly 1.4×10⁸platelets were collected into 200 ul of plasma and extracted using theprotocols described herein using either one single glass slides or twoseparate glass slides separated by at least 0.417 mm of air space (seeexample below). The total amounts of DNA recovered following elution at56° C. for 15 minutes is shown in FIG. 15. The amount of DNA purifiedfrom one glass slide is roughly one half of that purified from two glassslides in this experiment demonstrating quantitative recovery of DNAfrom 1.4×10⁸ platelets. Thus, the surface area of the glass slides usedfor the purification correlates directly to the total amount of DNArecovered from the lysates obtained using platelet concentrates.

FIG. 16 is a graph comparing the amount of DNA recovered from differentsamples as a function of the number of slides used to isolate DNA usingthe method of the invention. Referring to FIG. 16, the DNA bindingcapacity of the glass slides was analyzed using two types of lysates—arelatively DNA rich lysate (PC+Staphylococcus aureus as described below)and a relatively DNA poor lysate (PC alone as described below). 200 ulof platelet concentrates in plasma or 200 ul of platelet concentrates inplasma mixed with approximately 1×10⁹ cfu/ml Staphylococcus aureusbacterial cells were extracted using the protocols described herein. Thetotal amount of lysate was first dispersed in equal volumes across threesets of superimposed glass slides separated by 0.417 mm of air space(about 200 ul for each set of slides). Subsequently, the total lysateamount recovered from the first three sets of slides (about 600 ultotal) was pooled and spread across a fourth set of superimposed glassslides separated by 0.417 mm of air space. The DNA was recoveredfollowing overnight elution at room temperature in 200 ul of TE bufferfor each paired set of slides was quantified, and normalized to 200 ulvolume of lysate for each of the four slide sets. The total amount ofrecovered DNA from each set of slides in these experiments is shown inFIG. 16. This experiment shows that most of the DNA from the lysates isrecovered on the first three sets of glass slides and that the totalamount of DNA available for binding to the glass surface issignificantly less for the fourth glass slide. Moreover, the equal“levels” of binding of DNA in both types of lysate for the first threeslides in the DNA rich versus DNA poor lysates suggests that the glassslides function to bind a fixed percentage of the total DNA available inthe lysates that is independent of the total DNA concentration.Efficient recovery of even larger (microgram amounts) of DNA from oneset of paired glass slides in other experiments (data not shown) hasbeen observed, suggesting that the binding capacity of glass is largerwhen using biological materials than those used in these experiments.

To study the quality of the DNA extracted using the method of theinvention some of the DNA preparations were analyzed by real-timepolymerase chain reaction (RT-PCR) following purification from the glassslides. About one-one hundredth volume of the samples (about 2 ul)prepared from platelets ranging in number from 1.4×10⁸ to 3.5×10⁷ weresubjected to real-time PCR using the AmpliTaq Gold LD DNA polymerase(Applied Biosystems) as per manufacturer's instructions, except thatAmbion RT-PCR grade water was used throughout all points of the protocoland following purification of all reagents used in the PCR reactionsover a YM-100 ultrafiltration column (Millipore Inc.) as per themanufacturer's instructions for samples containing DNA. The PCR reactionwas followed in real-time using a MJ Mini Opticon instrument (BioRadInc.) by including SYBR Green dye (Invitrogen) into each PCR reactionmix at a concentration of 1× (diluted from 10000× stock using AmbionRT-PCR grade water). Amplification of a fragment of the HLA-DQA genefrom genomic DNA in leukocytes was carried out using primers asdescribed in Mohammadi et al., “Detection of Bacteria in PlateletConcentrates: Comparison of Broad-Range Real-Time 16s Rdna PolymeraseChain Reaction and Automated Culturing.” Transfusion 45: 731-736, 2005,incorporated herein by reference in its entirety. This showed that therewere no PCR inhibitors in the extracted DNA samples as well as templateconcentration dependence (as inferred from the various number ofplatelets and lymphocytes extracted) upon the C(t) values obtained fromthe reactions in FIG. 17. Threshold cycle C(t) reflects the cycle numberat which the fluorescence generated within a reaction crosses thethreshold and is inversely correlated to the logarithm of the initialcopy number. The C(t) value assigned to a particular well thus reflectsthe point during the reaction at which a sufficient number of ampliconshave accumulated. FIG. 18 graphs the C(t) values obtained versus theconcentration of platelets included in each DNA extraction.

Referring to FIG. 19, to demonstrate that the quality of the DNAextracted using the methods of the present invention are of sufficientquality to allow detection of bacterial DNA in platelet concentrates(PCs) some of the DNA preparations prepared on PCs contaminated withStaphylococcus aureus or Escherichia coli bacteria were analyzed byreal-time polymerase chain reaction (RT-PCR) following purification fromthe glass slides. About one-one hundredth volume of the samples (about 2ul) prepared from platelets and bacteria containing about 1×10⁹ cfu/mlof each type of bacteria were subjected to RT PCR using the AmpliTaqGold LD DNA polymerase (Applied Biosystems) as per manufacturer'sinstructions except that Ambion RT-PCR grade water was used throughoutall points of the protocol and following purification of all reagentsused in the PCR reactions over a YM-100 ultrafiltration column(Millipore Inc.) as per the manufacturer's instructions for samplescontaining DNA. The PCR reaction was followed in real-time using a MJMini Opticon instrument (BioRad Inc.) by including SYBR Green dye(Invitrogen) into each PCR reaction mix at a concentration of 1×(diluted from 10000× stock using Ambion RT-PCR grade water).Amplification of a fragment of the 16S rRNA gene from genomic DNA inbacteria was carried out using the universal 16S rRNA primers asdescribed in Mohammadi et al., “Detection of Bacteria in PlateletConcentrates: Comparison of Broad-Range Real-Time 16s Rdna PolymeraseChain Reaction and Automated Culturing.” Transfusion 45: 731-736, 2005,and is depicted in the graphs in FIG. 19.

Immobilized minor groove binders (e.g., bis-benzimide or BB dyes) can beused to detect DNA in a chamber of the device of the invention. BB dyeshave unique properties that may have advantages in PCR detection. Theblue fluorescence of the DNA bound BB dye (460 nm) is not read oncurrent commercial thermal cycling fluorimeters, but can be read on aseparate channel or in a separate instrument. This allows the DNAconcentration to be measured inside the card if BB dye is added to theelution buffer. The presence of the BB dye does not significantly affectthe reading of the standard green (520 nm) to red (650 nm) fluorescentdye used to quantify amplified nucleic acids. In one aspect of theinvention, a BB dye as a bulk DNA detecting dye is used to provideadditional control of the DNA extraction process. NAT reagents (primers,dNTPs, Taq polymerase) can be added to the elution buffer allowing forDNA amplification to take place in the vessel of the invention. When aBB dye is also present in the PCR mix, it can also be used to detect thepresence of amplified DNA.

As noted above, in one aspect, the invention provides methods forextracting and quantitating nucleic acids from biological samples, suchas blood and blood products, for the purpose of determining the presenceof microbial contaminants in the sample. In another aspect, theinvention provides methods that utilize the nucleic acid extractionmethod and further amplify the extracted nucleic acids to provide forthe identification of the amplified nucleic acids.

Automated techniques for use of PCR on blood samples generally requiretedious DNA extraction protocols to remove interfering substances in theplasma. The DNA extractions can use automated liquid handling systemsthat are expensive and difficult to maintain. In one aspect, theinvention provides a “lab-on-a-chip” device that combines cellularlysis, DNA extraction and purification, and measurement of extractedDNA. A representative device of the invention is illustrated in FIGS. 1and 20-22 and described in detail below. The device of the invention isa vessel for receiving and processing a biological sample as describedabove, and because of its structure and form (e.g., in one embodiment anine-layer laminate, see FIGS. 20A and 20B) is also referred to hereinas a “card.” The device allows sample entry, digestion, and DNApurification to take place inside a closed vessel that allows the lysis,binding and wash buffers to be delivered in a controlled fashion(aseptic sampling and handling). After capture in the purificationchamber of the device, the extracted and processed DNA can remain insidethe closed card for storage until ready for NAT.

In one embodiment of the method of the invention, the extracted DNA iseluted from the card using an elution buffer and transferred to acommercially available thermal cycling fluorimeter for quantitativeanalysis of the DNA by PCR.

In another embodiment, fluorogenic DNA binding dyes can be added to theelution buffer to measure the amount of purified DNA. The device of theinvention includes a window that allows for the determination of thequantity of purified DNA by fluorescence. The window allows forexcitation of the dyes and measurement of their emission to provide aquantitation of purified DNA. The inclusion of a blue fluorescent dye(e.g., a bis-benzimide, BB, having emission maximum at about 460 nm) inthe elution buffer allows the quantitation of extracted double strandedDNA (dsDNA) prior to NAT. The method provides a positive control for theDNA extraction process. Other conventional fluorescent materials (green,yellow, orange, red emissions) can be used with standard DNA probetechnology to detect primer directed amplification of target DNAfragments in real time.

To study utilize the blue fluorescent dye in real time PCR, a UVexcitation source/blue fluorescence detector instrument is required. Asuitable instrument is a thermal cycling fluorimeter having anLED/photodiode-based optical platform (BioRad Mini-Opticon). Inpractice, a filter set for SYBR green (DNA-bound form Ex=490 nm, Em=520nm), the commercial standard for DNA detecting fluorescent dyes, wasused.

The Mini-Opticon system noted above worked well for a PCR assay usingthe lymphocyte gene HLA-DQA1. Amplification of a fragment of the HLA-DQAgene from genomic DNA in leukocytes was carried out using primers asdescribed in Mohammadi et al., “Detection of Bacteria in PlateletConcentrates: Comparison of Broad-Range Real-Time 16s Rdna PolymeraseChain Reaction and Automated Culturing.” Transfusion 45: 731-736, 2005.The results of the assay showed that there were no PCR inhibitors in theextracted DNA samples using the glass slide method of the invention. Asexpected, C(t) values increased with decreasing template concentration.The Mini-Opticon cannot be easily adapted to read both green and bluefluorescence.

A second suitable thermal cycling fluorimeter is commercially availablefrom BioRad under the designation Chromo4. The fluorimeter houses acustomized photonics shuttle that moves an LED/photodiode housing tovarious positions over a 96-well plate. In practice, the shuttleincludes a UV-LED light source and suitable filters (Ex=360 nm, Em=460nm). Commercially available UV-LED sources have ideal wavelength forexcitation of the BB dye. In one embodiment, the invention provides athermal cycling fluorimeter capable of both green (labeled probe foramplified nucleic acid quantitation) and blue (dye for extracted nucleicacid quantitation) emission detection.

Thus, in another aspect of the invention, a method for amplifying andquantifying the amount of nucleic acids in a sample is provided. In oneembodiment, the method includes:

(a) introducing a sample containing cells in a liquid medium into afirst chamber of a vessel, the vessel comprising the first chamber and asecond chamber, wherein the first chamber is in liquid communicationwith the second chamber and wherein the second chamber has at least aportion of one surface effective for binding nucleic acids;

(b) lysing the cells to provide nucleic acids in the liquid medium;

(c) transferring at least a portion of the liquid medium from the firstchamber into the second chamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to a surface of the second chamber effective for bindingnucleic acids, to provide isolated nucleic acids;

(e) releasing the isolated nucleic acids from the surface of the secondchamber effective for binding nucleic acids by contacting the isolatednucleic acids with a buffer solution;

(f) treating the isolated nucleic acids with a nucleic acidamplification reaction mixture under conditions for amplifying theisolated nucleic acids to provide amplified nucleic acids;

(g) contacting the amplified nucleic acids with a fluorescent compoundhaving a fluorescence intensity dependent on the concentration ofnucleic acids; and

(h) measuring the fluorescence of the fluorescent compound to determinethe quantity of amplified nucleic acids.

In one embodiment the method includes repeating steps (f) and (h) apre-determined number of times to determine the amount of amplifiednucleic acids.

In another embodiment, the method includes contacting the isolatednucleic acids with a fluorescent compound having a fluorescenceintensity dependent on the concentration of nucleic acids and measuringthe fluorescence of the fluorescent compound to determine the quantityof isolated nucleic acids prior to step (f).

In one embodiment, the fluorescent compound is immobilized in the secondchamber. The fluorescent compound can be immobilized in the device asone or more patches of a film that can be adhered inside the secondchamber (e.g., the serpentine-shaped chamber or S-channel, see FIG. 1,reference numeral 400). Through the use of films of immobilizedfluorescent compounds, concentrations of DNA-detecting dye to be readilyadjusted.

The immobilization chemistry used to attach a representative DNA-sensingmaterial (e.g., BB-NH₂) to the glass slide surface can be varied. In oneembodiment, the DNA-sensing material is attached to human serum albumin(HSA) as a biocompatible linker to provide a sensor film. Albumin isoften used to coat surfaces to prevent DNA from adhering to them, adesirable feature in the sensor film. In one embodiment, a BB-NH₂ dye iscoupled to HSA in various dye to protein ratios via the carboxyl groupson the aspartic acid residues. Alternatively, an electrophilic form ofthe BB dye (such as the cyanuric acid derivative) can be attached to theHSA via the lysine residues. The ability of BB-HSA (in solution) toquantitate dsDNA can be optimized (loading levels, linker type). Thefluorophore/protein linkers and loading level can be tuned forfluorescent signal and dynamic range of response.

Non-porous polymeric surfaces functionalized with NHS ester groups mayalso be used for attaching the DNA-sensing material to provide the DNAsensing film. In this embodiment, the surface of non-porous polymericsubstrates can be directly functionalized for use in the DNA sensorfilm. In one embodiment, the substrate includes a polymeric polyethyleneglycol (PEG) surface. Various linkers can be introduced to the startingBB-NH2 dye by simple amide coupling chemistry. Scheme 1 belowillustrates a representative method for covalently coupling aDNA-sensing material to a polymeric substrate.

As shown in Scheme 1, the CH insertion of the photoactivated nitreneintermediate is adaptable to various polymeric substrates to providevarious thin film materials. Suitable substrates include polypropyleneand PVC, each having low binding to DNA.

Referring to Scheme 1, a derivative of the BB fluor that isphotoactivatable (PFPA-BB) is used for direct immobilization topolymeric substrates. In one embodiment, hydrophilic PEG linkers areincluded intermediate the PFPA and the BB fluor to allow conformationalflexibility of the BB dye and improve DNA binding efficiency.

In one embodiment of the immobilized DNA-sensing material, the sensorsubstrate includes a PFPA derivative of biotin with a PEG linker coatedon a polymer film, irradiated at 254 nm, and washed. The amount ofsurface bound biotin can be measured by treating withtetramethylrhodamine (TAMRA) labeled streptavidin (SA). Excess TAMRA-SAis washed off to measure 550 nm fluorescence of the biotin-SA complex.

In another embodiment, the DNA-sensing material is not immobilized, butrather introduced into the device of the invention so as to contact thenucleic acids to be quantified. In one embodiment, the nucleic acidamplification mixture includes a fluorescent compound.

In yet another embodiment, the buffer solution comprises the fluorescentcompound and the nucleic acid amplification reaction mixture.

In the methods above, the fluorescent compound is a minor groove binderor an intercalating agent. In one embodiment, the fluorescent compoundis a bis-benzimide (BB) compound (e.g., BB derivative).

In one embodiment, the method further includes determining the presenceof a gene or gene product in the sample by the presence of amplifiednucleic acids. As used herein, nucleic acid amplification reactionmixture refers to a mixture containing the components necessary foramplifying nucleic acids by PCR. In general, the nucleic acidamplification reaction mixture contains one or more primers that arecomplementary to the DNA region at the 5′ and 3′ end of the DNA regionthat is to be amplified; a DNA polymerase such as Taq polymerase;deoxynucleotide triphosphates (dNTPs); buffer solution; divalent cationssuch as magnesium (Mg²⁺) or manganese (Mn²⁺) ion; and monovalent cationssuch as potassium (K⁺) ions. The term “amplified nucleic acids” refersto nucleic acids produced in the method during the nucleic acidamplification step by reaction of the isolated nucleic acids with thenucleic acid amplification reaction mixture (e.g., by PCR).

The method may also include identifying a type of a gene or gene productin a sample by the presence of amplified nucleic acids. In thesemethods, specific nucleic acid probes that identify the gene or geneproduct are used. These probes are labeled with fluorescent compoundsand the specific gene or gene product is identified by fluorescencemeasurement.

In one embodiment, conditions for amplifying the isolated nucleic acidsare those of a polymerase chain reaction (PCR). In one embodiment of themethod, the isolated nucleic acids are transported from a proximal endof the second chamber to a distal end of the second chamber, theproximal end having a first temperature zone and the distal end having asecond temperature zone. The first temperature zone is at about 65° C.and the second temperature zone is at about 95° C. See, for example,FIGS. 22A and 22B.

When NAT reagents (e.g., PCR primers, Taq polymerase, dNTPs) are addedto the elution buffer, then analysis of specific genetic sequences canbe detected in the card by thermal cycling. Isothermal DNA or RNAamplification methods such as transcription-mediated amplification (TMA)can also be used in the methods of the invention.

For the methods of the invention that include nucleic acidamplification, conventional reagents and buffers are used. Suitablereagents are purified of endogenous and exogenous nucleic acidcontamination using one of several well known methods. It is well knownthat bacterial nucleic acids can contaminate aqueous solutions and thatsmall fragments of DNA can pass through 0.2 um filters used to removecontaminants. The magnitude of contamination when carrying outbroad-range rDNA PCR is highly variable and may be overestimated. Falsepositives are minimized by the methods and device of the invention. Taqpolymerase can be contaminated with nucleic acids from the bacterialspecies used for its manufacture. Ultrafiltration of aqueous reagentsare suitable for purification reagents useful in the methods of theinvention.

For extraction and elution of nucleic acids in the methods of theinvention, extraction and elution reagents and buffers are used. Theseinclude lysis buffer, proteinase K (or another protease), ethanol, Wash1 buffer, Wash 2 buffer, Elution buffer (±BB-NH2 dye) (±SYBR green), asdescribed below.

For the methods of the invention that include nucleic acid amplificationby PCR, conventional PCR reagents and buffers are used. Suitable PCRreagents include master mix including Mg²⁺ containing buffer, NTPs, Taqpolymerase, 16S rRNA gene primers, 16S rRNA gene probe (FAM label),HLA-DQA1primers, HLA DQA1probe (VIC label).

The methods and device of the invention can be used for quantitativePCR. The use of a blue fluorescent compound having a fluorescentintensity dependent on nucleic acid concentration (e.g., in theS-channel of the device) provides quality assurance of effective sampleacquisition and lysis. The inclusion of two temperature zones to thedistal ends of the glass walled S-channel allows for quantitative PCR inthe device. See, for example, FIGS. 22A and 22B, reference numerals 700and 800.

In practice, a PCR master mix with the appropriate fluorescent probe(s)and primers is added to the extracted DNA and the PCR mix (900 in FIGS.22A and 22B) is pumped back and forth between the two temperature zones(700 and 800 in FIGS. 22A and 22B). The method of the invention providesfor cycling of the PCR reaction mixture between the two temperaturezones and allows for rapid PCR. Heat transfer into the thin film of thePCR mixture (900) will be rapid as the mixture enters a each temperaturezone. A variety of fluorimeters can be utilized in the method andsystem.

As depicted in FIGS. 22A and 22B, a representative device can include atleast two constant temperature zones (700 and 800). Alternatively, theentire device can be subject to rapid cycling temperature of the smalland well insulated device.

The relatively small thermal mass of the PCR mix means that it willrapidly change temperature when moved from the lower to high temperaturezone. The lower temperature (700, about 65° C.) zone will act as a heatsink for the mixture after being pumped from the high temperature (800,about 95° C.) zone. If the surrounding insulation prevents excess heattransfer from the high to low zone, then again resistive heaters may beemployed. Without a means to remove heat from the lower temperatureregion, heat transfer through the continuous glass slides can raise the65° C. temperature zone above annealing temperature. Therefore, in oneembodiment, the low temperature heat source is a thermoelectric coolingdevice.

The device includes an optical path for the fluorometric quantitation ofnucleic acids. In one embodiment, the optical path includes a UV-LED onone side of the chamber and the 460 nm filter/photodiode on the otherside. The power/pulsing of the LED and the gain on the photodiode can beadjusted. Fluorescent signal is collected periodically to determine theposition of DNA-sensing material (e.g., BB) in the device and to measureincrease in DNA-dependent fluorescence as correlated with cycle number.The data for fluorescence and cycle number is collected on a computerand processed for q-PCR analysis. In one embodiment, the optical pathincludes the use of a probe (multiple fiber bundle including excitationand emission carrying fibers) that interrogates the PCR mix through awindow in the device (see FIG. 22B, reference numerals 1000 and 1100).

In certain embodiments, the methods include the use of a dye (e.g., blueemission) to quantitate extracted and isolated DNA prior toamplification. In one embodiment, the nucleic acid amplificationreaction mixture includes a first fluorescent compound having afluorescence intensity dependent on the concentration of isolatednucleic acids and a second fluorescent compound having a fluorescenceintensity dependent on the concentration of amplified nucleic acids. Thefirst fluorescent compound may be a minor groove binder or anintercalating agent, and the second fluorescent compound may be anoligonucleotide probe specific for a first gene or gene product.

The above methods, the nucleic acid amplification reaction mixture mayalso include a third fluorescent compound having a fluorescenceintensity dependent on the concentration of amplified nucleic acids. Inone embodiment, the third fluorescent compound is an oligonucleotideprobe specific for a second gene or gene product.

The methods noted above multiprobe PCR methods that are useful forsimultaneously detecting and speciating bacteria in clinical specimens(e.g., PRP).

Representative fluorimeters useful in eliciting and measuringfluorescence in accordance with the methods and devices of the inventioninclude commercially available instruments including the Bio-Tek PlateReader (end-point assays on a fluorescent plate reader using anautomated protocol and uniform reagents); the Ocean Optics (OO) fiberoptic fluorimeter (equipped with a UV-LED available from Nichia (Japan)as the excitation source having 360 nm emission that is an excellentspectral overlap for excitation of the bis-benzimide dyes); and BloodCell Storage Inc.'s two-color fiber optic fluorimeter (Seattle, Wash.),described in WO 06/023725, expressly incorporated herein by reference inits entirety. Two-color PCR with this device includes a fiber opticbundle and may be optionally configured with an LED on one side of thecard and a filtered photodiode on the other. The advantage of eitheroptical configuration is the ability to optimize LED light sources andfilters for each different DNA probe color of interest; reader filters(Ex=528 nm, Em=568 nm and 600 nm) have excellent spectral discriminationfor commercially available fluorescent DNA probes such as Yakima Yellow(Em=550 nm) and Redmond Red (Em=600 nm) commercially available from GlenResearch.

In the above methods the sample may be blood or a blood product (e.g.,PRP).

In another aspect of the invention, a fluidic system including a vesselis provided.

The vessel includes:

(a) a fluid inlet port;

(b) a first chamber in fluid communication with the inlet port;

(c) a second chamber in fluid communication with the first chamber,wherein the second chamber has at least one glass surface;

(d) a first channel that connects the first chamber and the secondchamber;

(e) a fluid outlet port; and

(f) a second channel that connects the second chamber to the fluidoutlet port.

In one embodiment, the second chamber includes two glass surfaces (i.e.,top and bottom). See FIGS. 1 and 20-22 illustrating a representativevessel of the invention, (reference numeral 450 refers to the glasssurface of second chamber 400).

The use of large glass surfaces for efficient capture of genomic DNAappears to have advantages over finely divided silica particles. Otherhigh surface area glass materials (membranes, diatomaceous earth,powdered glass, silica gel) have been used to get better DNA bindingefficiency. Although the DNA binding efficiency is high with thesematerials, the long DNA molecules get entrained in the silica andrecovery can be low. The resulting DNA is sheared during the processingsteps (vortexing) and these DNA fragments are recovered. Flat glasssurfaces, however, are efficient for capture of small amounts of DNAfrom dilute samples.

The vessel is preferably made from materials that exhibit lowautofluorescence and very low binding of DNA. Representative materialsinclude acrylic, polycarbonate, polypropylene, and polyvinylchloride,but not polystyrene. The materials should also be impervious to ethanol.Suitable materials include polypropylene (a low surface energy thinfilm); polyester terephthalate (PET); cast acrylic (a highmolecular-weight rigid material); 300LSE adhesive film (3M); 467MPadhesive film (3M); and Transil silicone adhesive film.

Suitable glass materials for the vessel include glass avalable instandard slide (25×75 mm) and cover slip (20 mm or 25 mm squares) andBorofloat (i.e., Pyrex) available in a variety of sizes: 100, 125, 150,200 mm diameter by 0.5 mm thick disks, 25, 50, 100, 150 mm squares by0.5 mm thick.

In one embodiment, the system include a pumping means. The pumping meanscan be effective for transporting fluids through the second chamber. Thepumping means can also be effective for transporting fluids from theinlet port to the fluid outlet port. With pumping a 20-200 ul bead ofelution buffer or water travels and down the S-channel to collect theDNA after purification at a temperature between 18-56° C. This allowsthe eluted DNA to be concentrated in the device.

In the device above, the first chamber has a surface comprising apolymeric covering. This covering allows an interface with a vibratingdevice (sonicator or piezobuzzer) to aid cell lysis. The thin coveringtransfers energy without causing leakage from the vessel. Duringfabrication, the polymeric covering should be sonicated in isopropylalcohol to remove combustion products of the laser-cutting process.After fabrication, the cards are preferably treated with ethylene oxideor gamma sterilization to remove competing pathogens. Off-card reagentspreferably pass a 2 micron cellulose filter on entry to removecontaminants. The reagent ports on the card provide an interface toyellow and blue pipette tips. A needle-septum interface can be provided.

In one embodiment, the second chamber comprises at least one immobilizedfluorescent compound having a fluorescence intensity dependent on theconcentration of nucleic acids. In this embodiment the second chambercan include a window through which the fluorescence intensity of thefluorescent compound can be measured. In one embodiment, the fluorescentcompound is immobilized in a region sufficiently proximate to the windowso as to allow fluorescent measurement. The fluorescent compound may bea minor groove binder or an intercalating agent. In one embodiment, thefluorescent compound is a bis-benzimide compound (e.g., hexylaminemodified bis-benzimide).

In one embodiment, the vessel is a component of a system that furtherincludes a device for eliciting and measuring the fluorescent intensityof the fluorescent compound(s). Suitable devices include the fluorimeternoted above.

Representative devices of the invention were fabricated for DNApurification in a microfluidic card format. FIG. 1 illustrates afabricated card. Glass slides are attached to the cards directly aboveand below the S-channel after the release layers are removed. The glassslides are aligned against the north edge and, once attached, sandwichthe S-channel between them. The rectangular cutouts are 200 um widerthan a standard glass slide. The combined layers are also illustratedschematically in FIGS. 1 and 20B, and the individual layers areillustrated in FIG. 20A. Primary features include two connector portslocated on layer 9, a triangular chamber formed by layers 2-6, aserpentine channel (S-channel) formed by layers 4-8, and an alignmentedge formed on the card bottom by layers 1-3 and on the card top bylayer 9. The two ports (inlet 100 and outlet 600) requiring fittings areat the top of the card. Standard size glass slides are placed inside thecutout rectangles sandwiching the serpentine channel (S-channel). SeeFIGS. 1 and 20B, reference numeral 450.

FIG. 1 is a schematic illustration of a representative fluidic device ofthe invention, or DNA card. Referring to FIG. 1, card 10 includes fluidinlet 100 into a first chamber 200. First chamber 200 is connected tosecond chamber 400 by first channel 300. Second chamber 400 includes aserpentine S-channel and at least one glass surface 450. The card can beassembled with glass slides on both sides of the S-channel. Secondchamber 400 is connected to fluid outlet 600 by second channel 500. Inone embodiment, the second chamber is defined by layers 4-8 shown inFIG. 20A (S-channel) and two glass slides (floor and ceiling, see FIG.20B).

FIG. 20A is an exploded view of a representative DNA card of theinvention. The DNA card is fabricated with nine layers. Referring toFIG. 20A, layers 2-6 include first chamber 200. Layers 4-8 includesecond chamber 400. Second chamber 400 has a serpentine S-channel, shownin layers 4-8. Layer 6 includes inlet 100 and first channel 300. Layer 8includes second channel 500 and outlet 600. Glass surface 450 ispositioned over the rectangular cut-out in layers 1-3, and a secondglass surface 450 is positioned over the rectangular cut-out in layer 9.FIG. 20B is an assembled view of a representative DNA card of theinvention.

There are several ways to input the sample and various wash buffers intothe vessel, or card. In the initial design stage Peek tubing stubs wereattached to the 9-layer laminated cards allowing manual input. Manualaddition allowed the various buffers to be optimized for volume,incubation time, and flow rate. Alternatively, standard 1 mlpolypropylene syringes or a programmable peristaltic pump can be usedwith tubing and Luer lock adaptors. The leak-proof property of the cardscan be advantageously used to aliquot reagents from a reservoir tube.

In one embodiment the positions of the inlet and outlet are on the upperedge of the card. This will allow multiple cards to be lined up in arack. The liquid lines will be connected to the tubing stubs using anO-ring seal to the pump head. A simple valve mechanism will switchbuffer sources and will be coordinated with the programmable pump.Computer-controlled 12-channel peristaltic pumps (Ismatec) that can beprogrammed to start/stop/change flow rate or reverse direction of flow.A rack of twelve cards would be of about the same dimensions as the12-channel pump. A separate Luer type port on the top side of the cardcan be added for addition of the sample. The (1 ml) syringe can remainin place during the entire lysis/DNA extraction/PCR process and bedisposed of with the card after disconnecting from the instrument.Leaving the syringe in place ensures maintenance of a closed system.

A batch of cards were fabricated by Aline Inc. (Redondo Beach, Calif.)and underwent the following treatment at their facilities prior toshipping: (1) post-fabrication the parts were sonicated in 20% aqueousisopropyl alcohol for twenty minutes; (2) a vacuum was applied (25 mmHg) and then isolated from the pump, after 16 hours, the parts did nothave moisture inside, and the vacuum had decreased to 18 mmHg, the partsalso developed more haze where the PSA degassed (typical behavior forthis class of PSA); (3) tube stubs were then attached and plasma was runthough the channel and chamber of each part, and the edges of theserpentine channel were also treated; and (4) the parts were then sealedinto foiled barrier bags with a heat seal.

FIGS. 21A-21F are schematic illustrations of the transport of fluidsthrough the fluidic device of the invention. Referring to FIG. 21A, card10 is shown with sample 25 in first chamber 200. After lysis andincubation card 10 is rotated clockwise 90 degrees and sample 25 istransported through the first channel 300 into the S-channel of secondchamber 400, as shown in FIG. 21B. DNA is captured on glass surface 450of second chamber 400, and chaotropic salt solution is introduced intocard 10 through outlet 600 (FIG. 21C). After washing card 10 is tippedagain to transport waste reagents back to first chamber 200, where theyare stored for disposal (FIGS. 21D-21F).

In a representative embodiment, the card is a ⅛ inch thick clear walledlaminated device with 2 internal chambers and 2 connecting channels. Thecard is the size of a standard 96 well plate. The S-channel has glasswalls formed by two glass microscope slides. Fluorescence can bemeasured in the S-channel using a standard plate reader. In use, 0.2 mlof sample is introduced through the inlet port (see FIG. 1, referencenumeral 100) and lysed in the triangular lysis chamber. After lysis, theS-channel is filled by gravity (no micro valves needed). DNA is capturedon the glass surfaces with high chaotropic salt. Wash buffers are pumpedinto outlet port inlet port (see FIG. 1, reference numeral 600) using aperistaltic pump or a syringe/pipetter. After washing, the DNA is elutedfrom the S-channel with low salt. FIG. 21 illustrates a gravity fillingmode of the S-channel. After returning to the starting position, wastereagents are pumped back into the lysis chamber (now waste chamber) forsafe disposal.

A more detailed description of a representative card and its featuresare as follows:

connecting channel widths=1.0 mm;

port diameter for all ports=1.0 mm;

S-channel fits within the area of a standard glass slide, width=25.3 mm,length=75.5 mm;

S-channel lines up with well positions of a standard 96 well microplate;

glass slide separation distance (glass chamber thickness)=22 mils (558.8um);

S-channel volume (glass chamber volume)=509 ul;

area covered by the S-channel is 910 mm², if both top and bottom glasssurfaces are included, the total glass area exposed to liquids=1820 mm²,which is approximately equivalent to the area of one surface of a singleglass slide (1910 mm²);

lysis chamber was designed to be separate from the glass chamber;

lysis chamber can be filled without allowing fluids to enter theS-channel;

lysis chamber volume≧815 ul;

lysis chamber is 1.27 mm thick.

exterior dimensions of the card without added fittings are approximately126.8 by 84.8 mm by 2.24 mm (thickness), with added tube stubs thicknessis approximately 10 mm.

The devices were assembled with glass microscope slides on both sides ofthe S-channel. The adhesive sealed well (no leaks with moderatepressure). The cards could be filled in a bubble free method by addingsolutions via the outlet port. By filling from the bottom up, displacedair is removed via the inlet port. As designed, the S-channel has avolume of about 0.6 ml. In practice, the card was placed flat on thebench and peristaltic pump was connected to the outlet port and the pumpwas used to successfully move a 0.075 ml sample back and forth in thechannel using a flow rate of 1 ml/min and manually reversing the pumpdirection.

FIGS. 22A and 22B are a schematic illustration of the device and methodof the invention useful for nucleic acid amplification. Referring toFIG. 22A, second chamber 400 has a proximal end with first temperaturezone 700 and a distal end with second temperature zone 800. Firsttemperature zone 700 is at about 65° C. and second temperature zone 800is at about 95° C. The first and second temperature zones are useful forthermal cycling for quantitative PCR in card 10. In one embodiment, anucleic acid amplification reaction mixture containing a fluorescentprobe and primers is added to isolated DNA in second chamber 400. Thenucleic acid amplification reaction mixture and DNA combination 900 istransported back and forth between first temperature zone 700 and secondtemperature zone 800. Referring to FIG. 22B, an appropriate wavelengthof excitation light is delivered with an LED and fluorescence intensityis detected with a photodiode housed in probe 1000. Fluorescence isdetected through window 1100 in first temperature zone 700.

In another aspect, the invention provides a method for determining themicrobial content of a blood product including:

(a) introducing a sample of a blood product containing cells in a liquidmedium into a first chamber of a vessel, the vessel comprising the firstchamber and a second chamber, wherein the first chamber is in liquidcommunication with the second chamber, and wherein the second chamberhas at least a portion of one surface effective for binding nucleicacids;

(b) contacting the cells in the liquid medium with a lysis buffer toprovide nucleic acids in the liquid medium;

(c) transporting at least a portion of the liquid medium into the secondchamber;

(d) extracting the nucleic acids from the liquid medium by binding thenucleic acids to the surface of the second chamber effective for bindingnucleic acids to provide isolated nucleic acids;

(e) releasing the isolated nucleic acids from the surface of the secondchamber effective for binding nucleic acids by contacting the isolatednucleic acids with an elution buffer to provide released nucleic acids;

(f) contacting the released nucleic acids with a nucleic acidamplification reaction mixture and a fluorescent compound having afluorescence intensity dependent on the concentration of nucleic acids;

(g) measuring the fluorescence of the fluorescent compound to determinethe quantity of released nucleic acids;

(h) treating the released nucleic acids and the nucleic acidamplification reaction mixture under conditions for amplifying theisolated nucleic acids by polymerase chain reaction to provide amplifiednucleic acids; and

(i) measuring the fluorescence of the fluorescent compound to determinethe quantity of amplified nucleic acids.

In one embodiment the method includes repeating steps (h) and (i) apre-determined number of times to determine the amount of amplifiednucleic acids.

In one embodiment, the method further includes contacting the isolatednucleic acids in the second chamber with a wash buffer and removing thewash buffer from the second chamber. The wash buffer comprises achaotropic salt solution.

In one embodiment, the fluorescent compound is immobilized in the secondchamber.

In another embodiment, the lysis buffer comprises a chaotropic saltsolution.

In yet another embodiment, the elution buffer comprises the fluorescentcompound and the nucleic acid amplification reaction mixture.

In the above methods, the fluorescent compound is a bis-benzimidecompound.

In one embodiment, the method further includes further incubating thesample for a pre-determined period of time prior to contacting the cellsin the liquid medium with the lysis buffer. The pre-determined period oftime can be 24 hours. For the specific application of the cards tobacterial contaminated platelet concentrate (PC), the sensitivity of thepublished PCR method is limited due to the small volume of platelet richplasma (0.2 ml). In addition, the published method only used 10% of theinitial extracted DNA from the PC. By combining culture amplificationwith PCR, a powerful bacterial detection technology can be created.Testing the freshly prepared PC after 24 hours of culture allows 1bacteria/ml at time of sampling (day 1=day of PC prep). The combinationof NAT and culture methods for sensitive detection of bacterialcontaminated platelets has not previously been described. A suitableseptum sealed aerobic culture bag (with media tablet) is sold by Pall (2ml PC volume) and septum sealed aerobic and anaerobic culture bottles(4-8 ml PC) are sold by BioMeriuex. The DNA card could also serve as asuitable culture vessel if desired. This would allow all operations(cell culture, cell lysis, DNA extraction, DNA purification, DNAanalysis, and NAT testing) to take place in a single, disposable “lab ona chip” design in accordance with the device and method of theinvention.

With microbial contamination levels at 1 cfu/ml, there is a statisticalprobability of 50% that a single bacterium would not be present in a 0.3ml sample. To circumvent this sampling issue, the device of theinvention can be used in combination with a platelet storage bag, asdescribed in WO 06/023725. A representative sample bag contains aculture broth pellet and is charged with 10 ml PRP. After 24 hours at37° C., a small sample (0.2 ml volume) is enriched in microbial DNA anda 0.2 ml sample size will contain the genomic DNA target. Transfer ofthis sample to the device of the invention allows for PCR detection ofthe captured genomic DNA target. By coupling microbial culture with PCR,the invention provides a sensitive microbial contamination test having areasonably short quarantine period for a day 1 test. The system ensuresthat no patient will receive a microbially-contaminated PC unit. The 24hour culture period fits well in the blood bank environment since mostPCs are transfused after 3 days of storage.

In another aspect the invention provides a method for quantifying andamplifying the amount of nucleic acids in a sample. The method includes:

(a) contacting nucleic acids from a sample with a nucleic acidamplification reaction mixture that comprises a first fluorescentcompound having a first fluorescence emission maximum and a secondfluorescent compound having a second fluorescence emission maximum,wherein the first and second fluorescence emission maxima are different,each having a fluorescence intensity dependent on the concentration ofnucleic acids;

(b) measuring the fluorescence of the first fluorescent compound todetermine the amount of nucleic acids;

(c) treating the nucleic acids and the nucleic acid amplificationreaction mixture under conditions for amplifying the nucleic acids toprovide amplified nucleic acids; and

(d) measuring the fluorescence of the second fluorescent compound todetermine the amount of amplified nucleic acids.

In one embodiment, the nucleic acid amplification reaction mixturefurther includes a third fluorescent compound having a thirdfluorescence emission maximum and a fluorescence intensity dependent onthe concentration of amplified nucleic acids, wherein the thirdfluorescence emission maximum is different from the first and secondfluorescence emission maxima.

In one embodiment, the second and third fluorescent compounds areoligonucleotide probes, each specific for a particular gene or geneproduct.

In another embodiment, the first fluorescent compound is a minor groovebinder or an intercalating agent. The first fluorescent compound can bea bis-benzimide compound, such as a hexylamine modified bis-benzimidecompound.

In another aspect, the invention provides a composition including anucleic acid amplification reaction mixture and a fluorescent compound.The fluorescent compound has a fluorescence intensity dependent on theconcentration of nucleic acids and has a fluorescent emission maximumless than about 500 nm. The emission wavelength less than about 500 nmassures that the blue emitting fluorescent compound can be quantitatedin the presence of other green and red emitting probes having emissionmaxima greater than about 500 nm. The composition is useful in methodsfor quantitating nucleic acids in the methods described above.

The following examples are provided for the purpose of illustrating, notlimiting the invention.

Examples Example 1 Lysis and DNA Extraction Using Glass Slides

200 ul of a sample such as blood, plasma, buffy coat, plateletconcentrates as well as any nucleic acid from the organism from whichthe sample is obtained in addition to any exogenous (bacterial, fungal,or parasitic) nucleic acids found in the sample is mixed with 200 ul ofa lysis buffer (described below) and subsequently incubated with 20 ulproteinase K of a specific activity of at 5-7.5 AU Anson Units at 56-65C for 15 minutes.

The source of the purified Proteinase K is the yeast Pichia pastoris asit would be predicted to contain fewer contaminating bacterial DNAmolecules which could be used to identify microbial contamination in thesample. A well known property of this enzyme is its activity across abroad range of pH (4-12.5) and an increase of its activity sevenfold inthe presence of SDS at temperatures ranging between 56° C. and 65° C.

The lysis buffer is composed of 1-3M GuSCN at a pH of 5-6.0 with 0.5 to1.2% Triton X-100 or mixtures of other detergents (such as SDS, NP40,CTAB, CHAPS, Sarkosyl, and Tween 20). Following this incubation thelysate is heated further to 95° C. for 2-10 minutes and then cooledslightly to a temperature of <65° C. The lysate is then subjected tomechanical disruption of the biological materials using a sonicatorprobe, or a piezobuzzer device, or another similar device capable ofintroducing high frequency resonant vibrations to the sample through alysis chamber wall of the DNA purification device (as described below)or directly by probe sonication at 350 MHz for 90 seconds (three 30second bursts) directly in an Eppendorf tube. The entire lysate issubsequently loaded onto a surface composed of two standard microscopeslides separated by a distance of at least 0.417 mm and incubated forabout 20-30 minutes. Following about 20 minutes of adsorption at atemperature between 18 and 60° C. the glass slides are washed with 500ul Buffer AW1 [pH ˜5.5 57% Ethanol] containing GuSCN. Subsequently 500ul of Buffer AW2 [pH 7.5 70% Ethanol] are added in order to rinse eachset of slides two times in successive order. Following the two washesthe slides are spun dry in an Allegra 6 (or similar) centrifuge at <2Kfor 10 minutes. Elution of the DNA is carried out next (200 ul) BufferTE [pH 8.5] or distilled water by incubating at a temperature between(18-70° C.) for 5-20 minutes, and then centrifuging in an Allegra 6centrifuge at <2K for 10 minutes.

Example 2 Basic Fluidic Protocol for the Use of a Representative Deviceof the Invention

A description of the use of a representative device of the invention, afluidics card, developed to facilitate the DNA extraction performance(sample is platelet concentrate (PC), a mixture of plasma with albumin,fibrinogen, a few leukocytes (1 M), and a high concentration ofplatelets (about 1×10⁸)) in a closed system environment is as follows:

a. Add 0.2 ml of PRP to the lysis chamber;

b. Add 0.2 ml of lysis buffer (with proteinase K or other protease) andmix (2 minutes);

c. Incubate at 65° C. (5 minutes);

d. Incubate lysis chamber at 95° C., then cool to <65° C., creating alower viscosity solution (2 minutes);

e. (Option 1) Further disrupt the biological materials using a devicecapable of introducing high frequency resonant vibrations to the samplethrough a lysis chamber wall (2 minutes);

f. Add 0.2 ml of ethanol and mix (1 minute), viscosity of solution issimilar to water;

g. Tip card to flow lysate into the glass-walled S-channel;

h. Incubate 5-30 minutes at ambient temperature;

i. Tip card back to start position;

j. Pump in 0.6 ml of wash #1 buffer (incubate 1 minute);

k. Pump in 0.6 ml of wash #2 buffer (incubate 1 minute);

l. Purge wash #2 from glass walled S-channel with air stream (1 minute);

m. Fill S-channel with 50-500 ul of elution buffer containing BB dye;

n. (Option 2) Pump elution buffer back and forth in S-channel (5minutes);

o. Read the dye fluorescence (460 nm) to quantitate DNA present;

p. Remove purified DNA solution from card and aliquot for PCR;

q. (Option 3—elution buffer contains PCR reagents) Pump PCR reagentsback and forth between 65° C. and 95° C. temp zones (5-30 minutes);

r. Read blue fluorescence at each PCR cycle (real-time PCR);

s. Analyzed data and quantitate microbial load (if any); and

t. Report results

The multiple processing steps point to the benefit of the lab-on-a-chipformat. A programmable pump and valve system can be used to add thevarious reagents to the card, and the incubation steps are controlled bythe position of the fluids in a thermally controlled instrument.Fluorescent reading of the DNA containing fluids in the card will bedirectly through the glass microscope slides themselves. Assuming 10minutes are needed for the DNA binding (step h), purified DNA (PCRready) can be isolated in 30 minutes. Some steps can also be shortenedor eliminated depending on the sensitivity requirements for microbialdetection. The quantitative PCR thermal cycling can be executed within30 minutes or less. The trim S-channel allows rapid temperatureequilibration and permit efficient primer extension.

1. A method for quantifying extracted nucleic acids, comprising: (a)eluting nucleic acids isolated on a solid phase with an elution bufferto provide released nucleic acids, the elution buffer comprising a firstfluorescent compound having a first fluorescence emission maximum and afluorescence intensity dependent on the concentration of nucleic acids;and (b) measuring the fluorescence of the first fluorescent compound todetermine the amount of released nucleic acids.
 2. The method of claim 1further comprising treating the released nucleic acids with a nucleicacid amplification reaction mixture under conditions for amplifyingnucleic acids to provide amplified nucleic acids, and measuring thefluorescence of the first fluorescent compound to determine the amountof amplified nucleic acids.
 3. The method of claim 1, wherein the firstfluorescent compound is a minor groove binder or an intercalating agent.4. The method of claim 1, wherein the first fluorescent compound has anemission maximum at a wavelength less than about 500 nm.
 5. The methodof claim 1, wherein the first fluorescent compound is a bis-benzamidecompound.
 6. The method of claim 1, wherein the first fluorescentcompound is ethidium bromide.
 7. The method of claim 1, wherein thefirst fluorescent compound is[2-[N-3-dimethylaminopropyl}-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium].8. The method of claim 2 further comprising contacting the amplifiednucleic acids with a second fluorescent compound having a secondfluorescence emission maximum and a fluorescence intensity dependent onthe concentration of amplified nucleic acids, wherein the first andsecond fluorescence emission maxima are different.
 9. The method ofclaim 8 further comprising measuring the fluorescence of the secondfluorescent compound to determine the presence of a gene or geneproduct.
 10. The method of claim 8, wherein the second fluorescentcompound is an oligonucleotide probe.
 11. The method of claim 8 furthercomprising contacting the amplified nucleic acids with a thirdfluorescent compound having a third fluorescence emission maximum and afluorescence intensity dependent on the concentration of nucleic acids,wherein the first, second, and second fluorescence emission maxima aredifferent.
 12. The method of claim 11 further comprising measuring thefluorescence of the third fluorescent compound to determine the presenceof a gene or gene product.
 13. The method of claim 11 wherein the thirdfluorescent compound is an oligonucleotide probe.
 14. The method ofclaim 2, wherein treating the released nucleic acids under conditionsfor amplifying the nucleic acids to provide amplified nucleic acidscomprises amplification using the polymerase chain reaction.
 15. Themethod of claim 2, wherein treating the released nucleic acids underconditions for amplifying the nucleic acids to provide amplified nucleicacids comprises amplification using isothermal DNA or RNA amplificationmethods.
 16. A method for quantifying and amplifying the amount ofnucleic acids in a sample, comprising: (a) contacting nucleic acids froma sample with a nucleic acid amplification reaction mixture comprising afirst fluorescent compound having a first fluorescence emission maximumand a second fluorescent compound having a second fluorescence emissionmaximum, wherein the first and second fluorescence emission maxima aredifferent, each having a fluorescence intensity dependent on theconcentration of nucleic acids; (b) measuring the fluorescence of thefirst fluorescent compound to determine the amount of nucleic acids; (c)treating the nucleic acids and the nucleic acid amplification reactionmixture under conditions for amplifying the nucleic acids to provideamplified nucleic acids; and (d) measuring the fluorescence of the firstfluorescent compound to determine the amount of amplified nucleic acids.17. The method of claim 16, wherein the first fluorescent compound is aminor groove binder or an intercalating agent.
 18. The method of claim16 further comprising measuring the fluorescence of the secondfluorescent compound to determine the presence of a gene or geneproduct.
 19. The method of claim 16, wherein the second fluorescentcompound is an oligonucleotide probe.
 20. The method of claim 16,wherein the nucleic acid amplification reaction mixture furthercomprises a third fluorescent compound having a third fluorescenceemission maximum and a fluorescence intensity dependent on theconcentration of amplified nucleic acids, wherein the third fluorescenceemission maximum is different from the first and second fluorescenceemission maxima.
 21. The method of claim 20 further comprising measuringthe fluorescence of the third fluorescent compound to determine thepresence of a gene or gene product.
 22. The method of claim 20 whereinthe third fluorescent compound is an oligonucleotide probe.
 23. A methodfor quantifying and amplifying the amount of nucleic acids in a sample,comprising: (a) contacting nucleic acids from a sample with a firstfluorescent compound having a first fluorescence emission maximum and afluorescence intensity dependent on the concentration of nucleic acids;(b) measuring the fluorescence of the first fluorescent compound todetermine the amount of nucleic acids; (c) treating the nucleic acidswith a nucleic acid amplification reaction mixture under conditions foramplifying the nucleic acids to provide amplified nucleic acids, whereinthe nucleic acid amplification reaction mixture comprises a secondfluorescent compound having a second fluorescence emission maximum and afluorescence intensity dependent on the concentration of nucleic acids,wherein the first and second fluorescence emission maximum aredifferent; and (d) measuring the fluorescence of the second fluorescentcompound to determine the presence of a gene or gene product.
 24. Themethod of claim 23 further comprising measuring the fluorescence of thefirst fluorescent compound to determine the amount of amplified nucleicacids.
 25. The method of claim 23, wherein the first fluorescentcompound is a minor groove binder or an intercalating agent.
 26. Themethod of claim 23, wherein the second fluorescent compound is anoligonucleotide probe.
 27. The method of claim 23, wherein the nucleicacid amplification reaction mixture further comprises a thirdfluorescent compound having a third fluorescence emission maximum and afluorescence intensity dependent on the concentration of nucleic acids,and wherein the first, second, and third fluorescence emission maximaare different.
 28. The method of claim 27, wherein the third fluorescentcompound is an oligonucleotide probe.
 29. The method of claim 27 furthercomprising measuring the fluorescence of the third fluorescent compoundto determine the presence of a gene or gene product.